Analytical method and apparatus

ABSTRACT

A method of examining thin layer structures on a surface for differences in respect of optical thickness, which method comprises the steps of: irradiating the surface with light so that the light is internally or externally reflected at the surface; imaging the reflected light on a first two-dimensional detector; sequentially or continuously scanning the incident angle and/or wavelength of the light over an angular and/or wavelength range; measuring the intensities of light reflected from different parts of the surface and impinging on different parts of the detector, at at least a number of incident angles and/or wavelengths, the intensity of light reflected from each part of the surface for each angle and/or wavelength depending on the optical thickness of the thin layer structure thereon; and determining from the detected light intensities at the different light incident angles and/or wavelengths an optical thickness image of the thin layer structures on the surface. According to the invention, part of the light reflected at said surface is detected on a second detector to determine the incident angle or wavelength of the polarized light irradiating the surface. An apparatus for carrying out the method is also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/234,829 filed Sep. 23, 2005, now U.S. Pat. No. 7,081,958; which is acontinuation of U.S. application Ser. No. 10/766,696 filed Jan. 27, 2004(U.S. Pat. No. 6,999,175); which is a continuation of U.S. applicationSer. No. 10/244,819 filed Sep. 16, 2002 (U.S. Pat. No. 6,714,303); whichis a continuation of U.S. application Ser. No. 09/368,461 filed Aug. 4,1999 (U.S. Pat. No. 6,493,097); which is a continuation of PCTApplication No. SE98/00196, filed Feb. 3, 1998; which claims priorityfrom Swedish Application No. 9700384-2, filed Feb. 4, 1997, all of whichapplications are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method and an apparatus for opticalsurface analysis of a sample area on a sensor surface.

2. Description of the Related Art

The interest for surface sensitive measuring techniques has increasedmarkedly recently as several optical techniques have been developed foridentifying and quantifying molecular interactions, which techniques donot require labelling. The most used optical technique so far is basedon surface plasmon resonance, hereinafter frequently referred to as SPR.

The phenomenon of surface plasmon resonance, or SPR, is well known. Inbrief, SPR is observed as a dip in intensity of light for a specificwavelength reflected at a specific angle (as measured by, e.g., aphotodetector) from the interface between an optically transparentmaterial, e.g., glass, and a thin metal film, usually silver or gold,and depends on among other factors the refractive index of the medium(e.g., a sample solution) close to the metal surface. A change of thereal part of the complex refractive index at the metal surface, such asby the adsorption or binding of material thereto, will cause acorresponding shift in the angle at which SPR occurs, the so-calledSPR-angle. For a specific angle of incidence, the SPR is observed as adip in intensity of light at a specific wavelength, a change in the realpart of the refractive index causing a corresponding shift in thewavelength at which SPR occurs.

To couple the light to the interface such that SPR arises, threealternative arrangements may be used, viz., either a metallizeddiffraction grating (see H. Raether in “Surface Polaritons”, Eds.Agranovich and Mills, North Holland Publ. Comp., Amsterdam, 1982), ametallized glass prism (Kretschmann configuration) or a prism in closecontact with a metallized surface on a glass substrate (Ottoconfiguration). In an SPR-based assay, for example, a ligand is bound tothe metal surface, and the interaction of this sensing surface with ananalyte in a solution in contact with the surface is monitored.

Originally, collimated light was used for measuring the SPR-angle, thesensing area being restricted to the intersection of the collimatedlight beam and the metal surface. The apparatus used was based onmechanical goniometry with two movable mechanical axes carrying theillumination and detecting components, the rotational center of whichwas placed in the center of the sensor area, i.e., one axis for theincident light and another axis for the reflected light which wasdetected by a single photodetector. A plane-sided coupling prism attotal internal reflection condition was used to preserve the collimatedbeam inside the prism, which, however, introduced a refraction atnon-orthogonal incidence at the prism, thereby introducing a beam-walkat the metal surface. A half-cylindrical coupling lens with orthogonalincidence of the optical axis of the light gave a fixed sensor area,however, introduced a beam-convergency, i.e., a non-collimated orquasi-collimated beam inside the prism.

In a development, the movable opto-mechanical axis on the illuminationside was eliminated by using a focused incident beam, so-called focusedattenuated total reflection (focused ATR), as described by Kretschmann,Optics Comm. 26 (1978) 41-44, the whole angular range simultaneouslyilluminating a focal line or point of a given sensing surface. The useof a detector matrix for detecting the reflected light eliminated themovable opto-mechanical axis also on the reflectance side, providing afaster SPR-detection than that of the prior art. Such systems aredescribed in, e.g., EP-A-305 109 and WO 90/05295. In the latter, lightbeams reflected from a specific sub-zone at the sensor area are imagedanamorphically so that beams in one plane (the sagittal plane) create areal image on a specific detector-pixel row of a matrix detector,permitting the occurrence of a local surface binding reaction to beidentified, while quantification of the reaction is obtained via angulardata for a reflectance curve measured along the same pixel row, wherethe reflectance curve is created by beams in a plane (the meridianplane) normal to the first-mentioned plane. Thus, one dimension of thematrix detector is used for real imaging simultaneously as the otherdimension is used for only angular measurement. This permits onlysub-zones arranged in a row to be simultaneously monitored and imaged.

In a variation of goniometry, the bulky mechanical axes were replaced byrotating or vibrating mirrors, respectively. A disadvantage of thatapproach is, however, that when scanning the incident angle by means ofa plane mirror, the point where the light beam hits the sensor surfacewill move along the internal reflection surface of the prism. Thisproblem was avoided by using a combination of rotating mirror andfocused SPR, e.g., as described by Oda, K., Optics Comm. 59 (1986) 361.In this construction, a first collimated beam of about 1 mm diameterimpinges on the rotational center of a rotating mirror placed at thefocal length of a focusing lens, thus producing a secondquasi-collimated beam, the distance of which to the optical axis dependson the reflecting angle of the mirror. The second collimated beam isfocused by a second focusing lens onto a prism base at total internalreflection conditions. During the rotation of the mirror, the angle ofincidence at the approximately fixed sensor area is scanned for thequasi-collimated beam.

Another approach to obtain a fixed and also enlarged sample spot isproposed by Lenferink et al., “An improved optical method for surfaceplasmon resonance experiments”, vol. B3 (1991) 261-265. This techniqueuses the combination of a collimated light beam illuminating a planerotating mirror, a focusing cylindrical (convex) lens after the mirrorand a half-cylinder lens for coupling the light to the sensing surface.By making the cylindrical lens focus the light on the focal surface ofthe coupling half-cylinder, using a relatively complex lens system, acollimated beam is obtained inside the coupling prism.

Other optical techniques similar to SPR are Brewster angle reflectometry(BAR) and critical angle reflectometry (CAR).

When light is incident at the boundary between two different transparentdielectric media, from the higher to the lower refractive index medium,the internal reflectance varies with angle of incidence for both the s-and p-polarized components. The reflected s-polarized componentincreases with the angle of incidence, and the p-polarized componentshows a minimum reflectance at a specific angle, the Brewster angle. Theangle at which both s- and p-polarized light is totally internallyreflected is defined as the critical angle. For all angles of incidencegreater than the critical angle, total internal reflection (TIR) occurs.

Schaaf et al., Langmuir, vol. 3 (1987) describes Brewster anglereflectometry using a micro-controlled rotation table and a movabledetector in scanning angle reflectometry around the internal Brewsterangle to study a protein (fibrinogen) at a silica/solution interface.The use of movable optomechanical axes and a rotation table gives a slowmeasuring procedure, and the sensor area is restricted to thecross-section of the collimated light beam with the sensor surface beinglimited by the need for non-beam-walking.

A focusing critical angle refractometer, based on a wedge of incidentlight which strikes the line of measurement, including the criticalangular interval to be measured, and which measures the one-dimensionalrefractive index profile along a focused line immediately adjacent tothe glass wall of a liquid container is described by Beach, K. W., etal., “A one-dimensional focusing critical angle refractometer for masstransfer studies”, Rev. Sci. Instrum., vol. 43, 1972. This technique islimited in that it enables only a one-dimensional sensor area, which isrestricted to the cross-section of the light line with the sensor area.

In all the above described prior art methods for reflectometricmeasurements, the reflectometric signal obtained represents an averagevalue for the sensor surface and the size of the sensor surface isrestricted, or minimized, to the extension of the collimated orquasi-collimated narrow beam, or focusing point or line. Therefore, suchSPR-based methods used to measure, for example, inter alia, proteininteractions are limited to quantitative information for sensing areaslocalized in one spot or one row of spots on the surface where aspecific interaction takes place. Approaches to monitor atwo-dimensional interaction pattern have been made for both macroscopicand microscopic SPR-based imaging of a sensing surface.

Thus, EP-A-341 928 discloses a method for monitoring a large SPR sensorarea in real-time by scanning a small focused beam, of, e.g., 10 μm, asa measuring sensing surface successively over the large area, morespecifically a DNA sequencing gel, for example 20×20 cm², thereby makingit possible to build up an image or picture of the sample distributionwithin the sequencing gel by means of a photodetector array. This methodrequires, however, the use of scanning mirrors for both addressing thesensor zones and scanning the angle and complex and expensive processingof angular and positional data from the mirror-scanners andphotodetectors, which limits the detection rate.

Yeatman and Ash, Electronic Letters 23 (1987) 1091-1092, and SPIE 897(1988) 100-107 disclose microscopic real imaging of the sensing surface,so called surface plasmon microscopy, or SPM. This was achieved by theuse of SPR in the Kretschmann configuration with a triangular prism forimaging dielectric patterns deposited on a silver film with a lateralresolution of about 25 μm, utilizing a focused beam in the form of aline scanned along the sensor surface. Also described is the use of anexpanded laser beam at the resonance angle to illuminate a larger areaand make a photograph of such an image by a positive lens in front ofthe photodetector. The illuminating beam is collimated and the angle ofincidence is adjusted by rotating the triangular prism. The method isproposed to be used for the examination of metal films, biological andother superimposed monolayers.

Image processing methods for such SPM using a collimated beam and a lensinserted into the reflected beam to create an image of the sampledistribution at the prism base are discussed in by Yeatman and Ash in“Computerized Surface Plasmon Microscopy”, SPIE, Vol. 1028 (1988) 231.

Okamoto and Yamaguchi have described a SPR-microscope wherein theposition of a collimated beam, SPIE vol. 1319 (1990) 472-473, or afocused beam, Optics Communications 93 (1992) 265-270, is scanned acrossthe sample surface in a Kretschmann configuration, the assembledpoint-SPR data thereby creating an image. In the focused beamalternative, a linear photodiode array is used for detection of theSPR-angle, in accordance with the principle of focused ATR as describedearlier by Kretschmann, supra.

Drawbacks with mechanically scanned SPR-sampling includes that a highlateral resolution and high speed for “real time monitoring” demands acomplex and expensive scanning mechanics (due to the bulkiness of theillumination and detection device).

EP-A-469377 describes an analytical system and method for thedetermination of an analyte in a liquid sample based on surface plasmonimaging. Surface plasmon images as a function of the angle of incidenceare monitored by a CCD-camera and analyzed by an image-software.Algorithms are used for comparing the measured SPR-angle for differentareas of the sensor surface for the purpose of eliminating thecontribution from a non-specific binding, and of calibration theresponse curve.

Rothenhausler and Knoll, Nature, 332 (1988) 615-617, demonstratessurface plasmon microscopy on organic films (a multilayer cadmiumarachidate), based on SPR in a Kretschmann configuration. A simple lensis used to form an image of the sample/metal interface. A collimatedbeam (plane waves) illumination and a movable bulky optomechanical axisare used to change the angle of incidence.

In the above prior art arrangements for SPM, the angle of incidence isvaried by the use of goniometry, either in a form where one or bothoptomechanical axes are moved (scanned) in relation to a fixed prism, orin a form using a movable optomechanical axis in combination with arotating prism, the common rotation point being in the center of theprism sensor surface, and the angle of incidence being derived from thechange in position or rotation of the mechanical axis throughmechanical, electronic, electromagnetic or optical means. It is readilyunderstood that such variation of the incident angle based on rotationof the mechanical axis carrying illuminating, imaging and detectormodules is rather slow and inaccurate if not a complex and expensivedesign is provided.

Another approach is disclosed by Kooyman and Krull, Langmuir 7 (1991)1506-1504, namely SPM using a small vibrating mirror in combination witha Kretschmann configuration to adjust the angle of incidence. Adisadvantage is, however, that the probed spot is not stationary duringthe angular scan, i.e., all sites within a sensing area are not probedby light at an equal angle of incidence range, unless bulky optics iscovering the sensing area at an excess.

Also microscopy based on Brewster angle measurements has been described.Henon and Meunier, Rev. Sci. Instrum., vol. 62 (1978) 936-939 disclosesthe use of a microscope at the Brewster angle for direct observation offirst-order phase transitions in monolayers. In this case, externalBrewster angle is measured, i.e., no internal reflection and no couplingprism, the camera plane being parallel with the sensor surface.

Similarly, Hönig and Mobius, J. Phys. Chem., vol. 95 (1991) 4590-4592describes the use of Brewster angle microscopy to study the air-waterinterface. Objects of about 3 μm diameter could be visualized by videorecording of p-polarized light reflected under a fixed Brewster anglefor the pure water surface.

A microscopic imaging ellipsometer has been described by Beaglehole, D.,Rev. Sci. Instrum., 59 (12) (1988) 2557-2559.

Multiple-angle evanescent wave ellipsometry, in the form of usingrotating optical means and a rotating prism for the variation ofincident angle, and a phase-modulated ellipsometer, has been used forstudying the polymer (polystyrene) concentration profile near aprism/liquid interface; see Kim, M. W., Macromolecules, 22, (1989)2682-2685. Furthermore, total internal reflection ellipsometry in theform of stationary optical means at a single angle of incidence has beensuggested for quantification of immunological reactions; see EP-A1-O 067 921 (1981), and EP-A1-0 278 577 (1988).

Azzam, R. M. A., Surface Science 56 (1976) 126-133, describes a use ofevanescent wave ellipsometry, wherein both the intensity andpolarization ellipse of the reflected beam can be monitored as functionsof the angle of incidence, wavelength or time. Under steady stateconditions, measurements as a function of wavelength and angle ofincidence can provide basic information on the molecular composition andorganization of the (biological) cell periphery. In a dynamictime-varying situation, measurements as a function of time can resolvethe kinetics of certain surface changes.

Abelès, F. et al., in Polaritons, Editor E. Burstein and F. De Martini,Pergamon Press, Inc., New York, 1974, 241-246, shows how extremelysensitive surface plasmons are to very fine modifications of thesurface, and the advantage of then measuring not only the reflectedamplitude, but also the ellipsometric parameters, amplitude and phase,of the reflected wave.

A focusing critical angle refractometer for measuring a one-dimensionalrefractive index profile being displayed in a graphic image for masstransfer studies has been described by Beach, K. W., The Review ofScientific Instruments, 43, No. 6 (1972) 925-928. Since this apparatususes one dimension in the image plane for a real image, and the otherdimension for projecting the reflectance versus angle of incidence, itcould not, however, provide a two-dimensional image of the refractiveindex distribution.

The prior art microscopy systems described above do not permit asufficiently rapid, sensitive and accurate scanning and measurement ofthe incident angle to permit highly quantitative multi-site real-timemonitoring of a sensor surface. Further, they are only suitable forimaging a limited sensor area of up to about 1×1 mm². They are also toolaborative and operator-dependent to be used in a commercial analyticalinstrument.

More particularly, the prior art apparatuses and systems for SPRmicroscopy and Brewster angle microscopy use either a high inertiascanner in the form of a goniometer where one or both mechanical axesfor the illumination side and the imaging side, respectively, arerotated in relation to a fixed prism, or a movable mechanical axis incombination with a rotating prism for scanning the optical axis, i.e.,the angle of incidence. In case the illumination consists of acollimated beam of light, the incident angle is measured directly on theangle steering signal without using the incident light, e.g., on thecontrol signal to the rotor-driving motor of a mirror, or by anelectronic or optoelectronic angle sensor placed on or at the rotatedaxis. Alternatively, a galvanometric or resonant low inertia scanner isused to drive a vibrating mirror to oscillate within a given calibratedangular range, determined by a given drive current, without monitoringthe actual incident angle of the light.

The necessary high refractometric sensitivity for determining therefractive index of the sensor surface with an apparatus constructedaccording to the above prior art in a long-term accurate commercialanalytical instrument could therefore only be achieved with a verycomplex and expensive design.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the abovedisadvantages and provide an optical method and apparatus that permitsmicroscopic as well as large area analysis of the sensing surface of asensor chip or plate with a high refractometric sensitivity and accuracyat high spatial resolution.

According to the present invention, the above object is obtained in alarge area or microscopy analytical apparatus of the above describedtypes, as well as in others, by using part of the angular orwavelength-scanned light that illuminates the sensor chip for monitoringand determining the momentary incident angle or wavelength of the light.

As will be further explained below, this enables an improvedquantitative measuring precision, accuracy and speed in thedetermination of the angle of incidence (alternatively wavelength)corresponding to the local reflection curve parameters, e.g.,reflectance minimum, at the surface structures on the sensor surface,such as surface-distributed reaction sites.

In one aspect, the present invention provides a method of examining thinlayer structures on a surface for differences in respect of opticalthickness, which method comprises:

irradiating the surface with light so that the light is internally orexternally reflected at the surface,

imaging the reflected light on a two-dimensional first photodetector,

sequentially or continuously scanning the incident angle and/orwavelength of the light over an angular and/or wavelength range,

measuring the intensities of light reflected from different parts of thesurface and impinging on different parts of the detector, at at least anumber of incident angles and/or wavelengths, the intensity of lightreflected from each part of the surface for each angle and/or wavelengthdepending on the optical thickness of the thin layer structure thereon,

and determining from the detected light intensities at each of thedifferent light incident angles and/or wavelengths an optical thicknessimage of the thin layer structures on the surface,

which method is characterized in that part of the light reflected at thesurface is detected on a second photodetector to monitor the incidentangle or wavelength of the polarized light irradiating the surface.

In another aspect, the present invention provides an apparatus forexamining thin layer structures on a surface for differences in respectof optical thickness, comprising:

a sensor unit having a sensing surface with a number of zones capable ofexhibiting thin layer structures of varying optical thickness,particularly as the result of contact with a sample,

a light source for emitting a beam of light,

optical means for coupling said light beam to said sensor unit,

first photodetector means,

means for sequentially or continuously scanning said light incident atsaid sensor surface over a range of incident angles and/or wavelengths,

means for imaging light reflected from said different parts of thesensor surface onto said first photodetector means for detecting theintensities of the reflected light,

means for determining each said angle and/or wavelength of lightimpinging on said sensing surface,

evaluation means for determining from the relationship between detectedintensity of the reflected light and incident light angle and/orwavelength, the optical thickness of each zone of said sensor surface tothereby produce and monitor a morphometric image of the opticalthickness of the sensor surface,

which apparatus is characterized in that it comprises secondphotodetector means, and that said means for determining said lightangles and/or wavelengths comprise means for focusing a part of saidlight reflected at the sensing surface onto said second photodetectormeans, wherein each position of said focused light on said secondphotodetector means is related to a specific angle and/or wavelength ofthe light incident at the sensing surface.

The term “optical thickness” as used herein defines a composite opticalproperty of a material that is a function of both its physical thicknessand its refractive index.

The term “surface structures” as used herein means any chemical,physical, biophysical and/or biochemical structures of thin or othertype on a sensor surface, especially structures produced via chemical orbiochemical interactions with species immobilized on the sensor surface.

It is understood that in accordance with the present invention, a seriesof images of a sensor surface are created, one image for each specificincident angle or wavelength. A part of the imaging detector, or aseparate detector, is used for measuring the incident angle orwavelength. This will be described in more detail further below.

The light is usually polarized prior to hitting the photodetector. Thismay be accomplished by using a light source which emits polarized lightor by placing a polarizer between the light source and the detector.

In one basic embodiment of the invention, monochromatic angularlyscanned light is used, and a main first part of the reflected collimatedbeam is used to produce a real image on a first main area of the matrixphotodetector, while a second part of the reflected collimated beam isfocused into a sharp line on a second specific minor linear area of thematrix photodetector. During the angular scan of the collimated beam thechange in angle of incidence is determined through the change inposition of either the maximum, or the centroid of the focused lightintensity detected by the detector elements along the linear detectorarea by use of an intensity-curve analysis algorithm.

In another basic embodiment of the invention, a scanned monochromaticlight at a fixed angle of incidence is used, and the second part of thereflected fixed collimated beam passes a wavelength dispersive element,and is then focused into a sharp point or line on a second specificminor linear area of said matrix detector. During the wavelength scan ofthe beam the change in wavelength is determined through the change inposition of either the maximum, or the centroid of the focused lightintensity detected by the detector elements along the linear detectorarea by use of an intensity-curve analysis algorithm.

In a preferred embodiment, the present invention provides an improvedreflectometric performance for an imaging method based on total internalreflection, such as e.g., surface plasmon resonance (SPR), or externalreflection, such as external Brewster angle spectroscopy orellipsometry, by the use of a novel optical design which will be furtherdescribed below and comprises a bifocal imaging system enablingsimultaneous sensor zone resolution and projection of rays formeasurement of the angle of incidence, and of the wavelength, on thesame matrix photodetector array, and a low inertia mirror scanner systemfor a mainly stationary illuminated area of the sensor chip incombination with a total internal reflection element, or a grating, forcoupling an evanescent wave to the sensor surface.

More particularly, such an apparatus may comprise an imaging systemhaving two different focal lengths in the same plane of projection(meridian plane), together with an identical focal length for thesagittal plane and the dominating part of the meridian plane, and acommon photodetector matrix array for image and angle monitoring.Preferably, the main part of the detector array lies in the real imageplane of the imaging system, while a minor part of the detector matrixarray lies in the back focal plane of the imaging system. Detectorelements within the real image plane monitor light intensity originatingfrom specific local spots at microscopic lateral resolution, while otherdetector elements within the back focal plane detect the position of thefocused line, i.e., the angle of incidence.

In an alternative form, the bifocal imaging enables detector elementswithin the real image plane to monitor light intensity originating froma specific local spot at microscopic lateral resolution, while otherdetector elements within the back focal plane detect the position of thefocused line corresponding to the wavelength.

The reflected light intensity varies with the angle of incidence, mainlydue to the angle dependent reflection coefficient, but may also vary dueto a movement of the illuminated area if the light intensity across theilluminating beam is not constant. By use 15 of an approximatelystationary illuminated sensor area, together with an image-intensitynormalization algorithm, a highly resolved light intensity of the localreflectance curves may be detected, which enables a high resolution inthe determined angle/wavelength at the corresponding reflectance minimaor maxima or centroid.

While it is preferred that the same detector array component is used forboth image detection and angle or wavelength detection, separatedetectors may, of course, also be used.

When both incident angle and wavelength of the light are to be measured,the detector may be divided into two (smaller) parts, and a larger part,wherein one of the smaller parts is used for detection of the incidentangle, and the other smaller part is used for detection of thewavelength, and the larger detector part is used for imaging.

As stated above, the present invention is characterized by an integraldetection of both image and incident light angle and/or wavelength toobtain a better accuracy and sensitivity in the multi-zone reflectometryand a faster analysis procedure than those obtained in the prior art.Thus, according to the present invention, the same collimated light isused (i) to illuminate each point on the sensor surface with apractically identical angle of the incident light, permitting a minimumdeviation (a maximum deviation of 0.002°) within the whole sensor areafor a large angular/refractometric dynamic range, and (ii) to measurechanges in this angle for each zone with a resolution of the order of0.0001°.

Thus, by integrating into the structure (i) a detector for a highlyresolved image of, for example, a multi-zone sensor surface, and (ii) adetector for the angle and/or wavelength, respectively of the incidentlight, and thereby reduce the sources of error for the measurement ofangle and wavelength, respectively, and their relation to a specificsensor subzone representation on the photodetector matrix, the followingadvantages may be obtained in relation to the prior art apparatusconstructions:

-   A higher resolution of absolute and relative reflectance minimum    angle,-   a higher resolution of absolute and relative reflectance minimum    wavelength,-   a higher image contrast and resolution of image contrast,-   a higher frequency for reflectance image data detection,-   a higher optomechanical robustness (less wear, lower maintenance    cost),-   a smaller total volume for the optomechanical apparatus, which    permits an improved (cheaper, quicker) thermostatting, and thereby    improved measuring system performance.

The apparatus according to the invention may use any of a number ofoptical principles for the scanning of the angle of incidence of thecollimated beam, known to those skilled in art, including the principlesdescribed above in the description of the prior art, as long as the beamdeflecting principle selected provides an angularly scanned collimatedbeam incident on a sensor surface at total internal reflectionconditions, in either a Kretschmann or Otto prism configuration, or agrating coupling configuration, during the probing of a mainlystationary area.

Exemplary such scanning principles are reflective beam steering throughmirrors or reflection diffraction gratings, and transmissive beamsteering through refracting scanners, or through transmissiondiffraction gratings or transient gratings (acousto-optical scanning).These beam-steering principles may be combined into suitable scannerconfigurations known to a person skilled in the art.

Likewise, the optical system principle for the determination ofreflection versus angle and/or wavelength of incidence may be selectedfrom a number of optical principles known to a person skilled in art,including those described above in the description of the prior art.Exemplary such optical principles are surface plasmon resonance (SPR),Brewster angle (both internal and external), ellipsometry angle (bothinternal and external), critical angle, and frustrated total reflectionwaveguide resonance.

In the case of also an evanescent wave absorbing sample interaction withthe sensing zone, the change in the complex refractive index correspondsnot only to a shift in the SPR-angle or SPR-wavelength (related to thechanged real part of the complex refractive index) but also to a shiftin depth of the intensity at the resonance.

For sensor applications involving absorbance, the apparatus according tothe invention may operate for reflectance-absorption imaging.

In the case of the incident light being coherent, a spatiallyheterogeneous sensing surface in terms of optical thickness orrefractive index modulates the relative phase of the evanescent wave,creating a stationary interference pattern in the image or real imageobtained. The incident light may after the total internal reflectioninterfere with light partially reflected at another interface of thesensor unit, e.g., on the opposite side to the sensor surface substrateinterface on the substrate. When, for example, a sample molecule isspecifically bound to certain areas within this sensing surface, theinterference fringes move into new positions. By tracking the positionof specific fringes by use of image process software, in relation to thescanned angle or wavelength of incidence, the local surfaceconcentration changes may be interferometrically monitored by theapparatus according to the invention. The interferometric feature ofphase-sensitive evanescent wave detection may increase therefractometric sensitivity in, for example, interferometric surfaceplasmon resonance image analysis, or interferometric Brewster angle andcritical angle image analysis.

Generally, the present invention creates an optically densitometrically(refractometrically) quantitated image of a sensing surface pattern inapproximative real-time by momentarily detecting the polarized lightreflectance of the sensing surface. While the reflectance in the sensingsurface preferably is internal, i.e., the light enters from a dielectricmedium of a higher refractive index towards a dielectric medium of alower refractive index, the reflectance may also be external, i.e., theprobing light passes through a layer, such as a sample layer, to beprobed on the sensor surface, e.g., external Brewster anglereflectometry and external ellipsometry.

Hereinafter, the present invention will be described in more detail, byway of example only, with regard to some embodiments of the invention.Reference is made to the accompanying drawings, wherein identical partsthroughout the Figures are indicated by the same reference designations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic meridian view of an embodiment of an opticalsensor apparatus and light ray paths thereof according to the presentinvention, which embodiment is based on scanning of the angle of theincident light.

FIG. 2 is another, parallel schematic meridian view of the embodimentshown in FIG. 1.

FIG. 3 is a schematic enlarged meridian view of a part of the embodimentshown in FIG. 1.

FIG. 4 is a schematic enlarged meridian view of a part of the embodimentshown in FIG. 2.

FIG. 5 is a schematic enlarged sagittal view of a part of the embodimentshown in FIG. 2.

FIG. 6 is a schematic sagittal view of a modified design of theembodiment shown in FIG. 5.

FIG. 7 is a schematic meridian view of the embodiment shown in FIG. 6.

FIG. 8 is a schematic perspective view of the embodiment shown in FIGS.3, 4 and 5.

FIG. 9 is a schematic perspective view of the embodiment shown in FIG.6.

FIG. 10 is a schematic perspective view of a modified design of theembodiment shown in FIG. 7.

FIG. 11 is a schematic exploded view of an alternative embodiment of anoptical sensor apparatus and light ray paths thereof according to thepresent invention, which embodiment is based on scanning of thewavelength of the incident light.

FIG. 12 is a schematic enlarged meridian view of a part of a modifiedversion of the embodiment shown in FIG. 11.

FIG. 13 a schematic sagittal view of the embodiment shown in FIG. 12.

FIG. 14 is a schematic meridian view of an alternative embodiment tothat shown in FIGS. 12 and 13.

FIG. 15 is a schematic sagittal view of the embodiment shown in FIG. 14.

FIG. 16 is a schematic exploded meridian view of an alternativeembodiment of the present invention based on ellipsometry.

FIG. 17 is another, parallel schematic meridian view of the embodimentshown in FIG. 16.

FIGS. 18 to 22 are schematic illustrations of prior art means useful forscanning the angle of incident light in apparatuses of the presentinvention.

FIG. 23 is a schematic meridian view of a modified design of theembodiment shown in FIG. 2.

FIGS. 24 a to 24 d are SPR-images of a sensor surface.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention relates to an optical methodand apparatus for large area or microscopical analysis of structures ona sensor surface, such as real-time monitoring of a chemical sensor orbiosensor surface. Usually, the sensor surface has a plurality ofindividual subzones or areas at which different interactions may takeplace and produce thin layer structures of optical thicknesses thatdiffer between the subzones. Such “multi-spot” surfaces may be used fora variety of analytical purposes. For example, a surface supportingdifferent ligands on the surface may be subjected to a sample that maycontain one or more species, or analytes, capable of binding torespective ligands on the surface. Thereby a sample may be analyzed forthe presence of several analyses “in one shot”. Other examples and usesare readily apparent to the skilled person.

According to the invention, the sensor surface in question is subjectedto illumination with, usually, collimated light on a mainly constantilluminated area and area position, and both image and angle ofincidence, and in an alternative form where more than one wavelength issequentially incident, also wavelength, are simultaneously monitored byuse of the same or another multichannel photodetector or matrixphotodetector array than that used for the imaging. A characteristicfeature of the invention is that a (usually minor) part of the angularor wavelength-scanned light that illuminates the sensor area for imagingpurposes is used to determine the momentary incident angle or wavelengthof the light. In that way, the momentary incident light angle orwavelength may be directly correlated with the simultaneously producedmomentary image of the sensor surface, permitting convenientquantification of the surface subzones where, e.g., a surface bindinginteraction has taken or takes place.

FIG. 1 schematically shows a total internal reflection-based embodimentof optical system and ray path according to the present invention, whichfor the purpose of illustration only is assumed to be a biosensor, forexample, of the type described in U.S. Pat. No. 5,313,264 and inJonsson, U., et al., BioTechniques 11:620-627 (1991).

In the system of FIG. 1, a light source, LS, illuminates a collimatoroptics, CO, to produce a parallel light beam which passes aninterference filter, I, as a monochromatic beam and impinges on a firstflat scanner mirror, SM1 to be deflected onto a second scanning mirror,SM2. The latter deflects the beam into a means for coupling the light toa sensor surface, SS. In the illustrated case, this coupling means is aprism, Pr, but may also be a grating as is well known in the art.

The sensor surface may be any of a variety of sensor surfaces (which areknown or will be known in the future) and, while its basic materialstructure largely depends on the optical detection principle used, itusually comprises an outer reactive layer, for example having differentspecific reactive ligands, e.g., antibodies, antigens, nucleic acidfragments, cells or cell fragments, etc., immobilized on definedsubzones. An exemplary biosensor element for SPR is described inEP-A-442922, whereas exemplary reactive biosensor surfaces are describedin U.S. Pat. No. 5,242,828 and U.S. Pat. No. 5,436,161. The lattercomprise a hydrogel (for SPR purposes bound to a gold or silver film),such as dextran, which supports or is capable of covalently bindingligands for reaction with sample analytes.

The sensor surface may further be based on biophysical reactions, wherethe specific sample interaction results in a structural changemanifested as a change in the thickness/refractive index distribution,including absorption, of the sensor layer. Such biophysical interactionsinclude, for example, protein crystallization, formation of proteinaggregates caused by precipitation, agglutination or flocculationreactions; the adhesion of membrane complexes, such as biological cellsand cell membrane fragments. For further details on the detection ofbiophysical interactions on a sensor surface it is referred to ourcopending international application PCT/SE96/01074.

While the sensor surface may be constructed directly on prism Pr in FIG.1, the system described in U.S. Pat. No. 5,313,264, referred to above,uses an elastic optical interface (“optointerface”) for coupling thelight between the prism and the sensor surface. Exemplary opticalinterface designs are described in U.S. Pat. No. 5,164,589 and ourcopending international application No. PCT/SE96/01522.

The sensor surface is (in the illustrated case) assumed to be exposed onits upper side to a sample containing analyses. The sample mayadvantageously be contacted with the sample in a flow type cell, e.g.,as described in the aforementioned U.S. Pat. No. 5,313,264 where one ormore flow cells are defined by the sensor surface being docked againstone or more open channels in a fluidic block or cartridge.

With reference again to the ray path in FIG. 1, the beam is totallyinternally reflected at the sensor interface side of the coupling prism.The p-polarized component of the beam then passes a polarizer, P, and amain first part of the beam is directed into a first main part of anobjective, which main part consists of a spherical objective, SO,producing a real image on matrix detector array, D, of the lightintensity reflected from the sensor surface area. The detector array Dis arranged such that the real image of the sensor area is produced on afirst rectangular main part, D1, of the array. That is, the first part,SO, of the objective has its real image plane positioned at the plane ofthe photodetector array. FIGS. 1, 2 and 3 at the detector array D denotethe respective images of the corresponding subzones on the sensorsurface SS, indicated at 1′, 2′ and 3′, respectively.

The two scanning mirrors, SM1 and SM2, have a related rotationalmovement which produces an angularly scanned collimated beam incidentwithin a range of angles of incidence on the sensor surface side of theprism Pr. As is readily appreciated by the skilled person, the “beamwalk” of the illuminated area may be reduced by a suitable choice of thedistances between mirrors and prism, and scanned angular range of themirrors.

Whereas only the above-mentioned first part of the objective is shown inFIG. 1, the remaining or second part of it is shown in FIG. 2, which isa meridian view of the optical biosensor system parallel to that in FIG.1 and illustrates how the ray path for a second minor part of the beamis directed into the second, minor part of the objective consisting ofthe spherical objective SO, mentioned above, combined with twocylindrical lenses, CL1 and CL2. The latter combination creates aprojection of these rays so that the collimated beam reflected at angleswithin the scanned range is focused onto a second linear minor part D2of the detector area, separated from the detector area used for realimage monitoring, so that each angle of reflectance corresponds to aspecific detector position within this detector area. That is, thesecond part of the objective has its back focal plane positioned at theplane of the photodetector array. The letters a, b and c at the detectorarray D2 indicate the positions on the back focal plane of the focusedbeams a′, b′ and c′, respectively, which exit the prism Pr, the threedifferent angles sequentially illuminating the whole sensor surface andrepresenting three different successive angles of the incident lightbeam.

The meridian bifocal imaging system described above (one focal lengthfor monitoring the real image, another focal length for monitoring theangle of incidence) enables a simultaneous monitoring of both theposition of a reaction site within the sensor area (real imaging), andof the quantitative measure of the amount of reacting species (analytes)at the site, by use of the same detector array.

In FIGS. 1 and 2, the inclined detector plane and the inclined couplingprism exit (non-orthogonal ray-passage at the exit of the prism)indicate the principle, known to anyone skilled in art, to reduce thedefocusing of the image at inclined imaging.

As already mentioned above, the coupling prism Pr may be replaced by acoupling grating (see, e.g., WO 88/07202 and PCT/GB91/01573).

A more detailed description of the coupling prism, sensor surface, andbifocal imaging system for a combined image- and angle monitoring at thesame detector array is shown in FIGS. 3 to 10.

FIG. 3 (which corresponds to FIG. 1) shows a meridian section of raybundles reflected at different points or sensor zone heights of thesensor surface, indicated at 1′, 2′ and 3′. Sensor zone heights 1′, 2′and 3′ are sharply imaged by the spherical objective SO at meridionalheights 1, 2 and 3 on the real image plane on the detector array. Forthese rays passing only the spherical objective SO, the detector planeis positioned at the real image plane of the lens system.

FIG. 4 (which corresponds to FIG. 2) shows a meridian section of raybundles reflected at different angles a′, b′, and c′, at the sensorsurface sagittal height 0′ (see FIG. 5). Rays reflected at each specificangle are sharply focused to a line at meridional positions a, b, and c,on the detector D2, by the combined spherical objective and cylindricalelements. For a specific angle of incidence of the collimated light, allthe rays will be focused to a line across the columns of detectorelement designated to angular monitoring. By determining the position ofthis light intensity peak along such a column, using a suitablealgorithm and an angular calibration procedure, a real-time measurementof the angle of incidence for the related sensor surface 2-dimensionalimage is enabled with high resolution, accuracy and speed.

Depending on the chosen angular range, an aperture stop, Ap, may be usedto select the rays, and thereby the length along the surface sagittalheight 0′, required for angular detection.

By introducing an obscuration in a part of the collimated beam, anaperture can be formed in the obscuration for passing of rays used formonitoring of angle of incidence, whereby the width and length of thefocused spot or line of these rays at the back focal plane can besuitably adjusted in relation to the size of the pixel and thepixel-separation in the pixel-array of the photodetector, or in the caseof a spot-position sensitive large area sensor, in relation to its area.As such, the number of pixels, or the area of the photodetector, coveredby the spot-intensity peak-width can be optimized for providing a highspot-position resolution, enabling a high sensitivity of measured angleof incidence. As described in FIGS. 2, 4 and 5, this collimated beampassing the aperture is internally reflected at 0′, passes lenses CL1and CL2, and meets the detector plane D2 at 0.

The obscuration may be made of any mainly non-transparent material ofsuitable opto-mechanical properties. The obscuration may be positionedat or within the illumination system, or between the illumination systemand the first scanning component. In a preferred embodiment, saidobscuration is positioned at the interference filter I, as illustratedin FIG. 23.

The aperture is an unobscured opening, taking the shape of a circle,quadrant, or rectangle. The aperture may be positioned either within, orat the edge of the obscuration.

In a preferred embodiment, the aperture is co-centric with the center ofthe illumination system in the plane of incidence, as shown in FIG. 23,while having its center decentered in relation to the illuminationsystem in the direction orthogonal to the plane of incidence, so thatthe obscuration covers a minor area of the collimated beam that leavesthe lens system CO, and the image of the obscuration covers 2-10% of thecommon photodetector area.

The aperture, has a typical width, in the direction of the plane ofincidence, ranging from 0.05-1 mm, a preferred width being 0.2-0.5 mm.In case of the aperture taking the form of a rectangular slit, the slitlength is typically within 0.5-3 mm, a preferred length being 1-2 mm.

A suitable masking of the sensor surface at sagittal height 0′ to limitthe reflection area, and/or a suitable structure that always gives totalreflection at the sagittal height part 0′ of the sensor surface may beused to obtain the necessary stability of the intensity peak used forangle determination. This may, for example, be accomplished by a sensorsurface which in addition to reactant zones also comprises a part (0′)which exhibits a mainly constant reflectance independently of incidentangle and wavelength and which thus, in for example SPR, does not giverise to a resonance.

A sagittal section of ray bundles reflected at different parts of thesensor surface is illustrated in FIG. 5. Sensor zone heights 4′, 5′, 6′,and 7′ are sharply imaged by the spherical objective at sagittal heights4, 5, 6, and 7 at the detector plane, D, while rays reflected atsagittal height 0′ also pass two cylindrical lens elements, CL1 and CL2,which create a defocused intersection of the rays at the detector planeat sagittal position 0. A detector window is indicated at W.

The two cylindrical lens elements may, in the sagittal plane, have asuitable radius, R, providing a refraction in order to locate the imageplane of the rays in front of the detector plane, thereby creating adefocusing of the rays across several detector columns used for theangular detection.

FIG. 6 shows a sagittal section of an alternative optical system design,wherein the rays used for measuring the angle of incidence are given asuitable ray path folding via two fixed mirrors M1 and M2.

FIG. 7 shows a meridian section of another optical design, similar tothat in FIG. 6, which provides for the ray path folding via two fixedmirrors M1 and M2 in order to obtain a required degree of overlap of theback focal plane and the detector plane within a large angular dynamicrange.

The objective and detector design shown in FIGS. 3 to 5 is schematicallyrepresented in a perspective view in FIG. 8. Corresponding (schematic)perspective views of the designs shown in FIGS. 6 and 7 are illustratedin FIGS. 9 and 10, respectively. Thus, the optical function ofmonitoring both the real image and the angle of incidence at the samedetector array, by use of the above-mentioned first and second parts ofthe objective, respectively, is depicted in FIG. 8, whereas FIG. 9depicts the beam deflection by the folding mirrors M1 and M2 inaccordance with the design shown in sagittal view in FIG. 6, and inmeridian view in FIG. 7.

An alternative optical biosensor system according to the presentinvention based upon wavelength scanning rather than scanning of theincident light angle is shown in FIG. 11. To this end the systemcomprises a scanning monochromator light source, and a wavelengthmonitoring device.

As previously, the biosensor system comprises a light source, LS, and acollimator optics, CO, to produce a parallel beam, which passes amonochromator, M, and is then directed into a coupling prism Pr (gratingcoupling is also possible). The collimated beam at a fixed angle ofincidence is totally internally reflected at the sensor interface sideof the coupling prism. The p-polarized component of the beam then passesa polarizer, P, and is cylindrically focused by lenses, L3 onto a slit,S.

As in the embodiments based on angular scanning above, a minor part ofthe beam is directed into the minor part of an objective, which minorpart consists of a spherical objective, SO, combined with cylindricallenses, L3 and L4, which create a collimated beam impinging on atransmission grating, T.

The light beam is then dispersed by the grating T so that the directionof propagation of the collimated beam depends upon its wavelength. Thisbeam is then brought to a focus by a cylindrical lens, L5(alternatively, a mirror in a folded configuration), so that for ascanned wavelength, a spectrum consisting of a series of monochromaticimages, λ1, λ2, and λ3 of the entrance slit S at the above-mentionedlinear minor area part of a two-dimensional detector array, D2, so thateach reflected wavelength corresponds to a specific detector positionwithin this detector area.

For a specific wavelength of incidence of the collimated light, all thedispersed rays will be focused to a line across the columns of detectorelements designated to wavelength monitoring. By determining theposition of the light intensity peak along such a column, using asuitable algorithm and a wavelength calibration procedure, a real-timemeasurement of the wavelength of incidence for the related sensorsurface (2-D) image is enabled with high accuracy and sensitivity.

As described above for the embodiments using angular scan, one may use asuitable masking at one side of the sensor surface (0′ in FIG. 13) tolimit the reflection area, and/or a suitable, always totally reflectingstructure at that sensor surface part to obtain a necessary constancy ofthe intensity peak used for wavelength determination.

In accordance with the description above for the embodiments usingangular scanning, one may also introduce an obscuration in a part of thecollimated beam, wherein an aperture can be formed in the obscurationfor passing of rays used for monitoring of wavelength of incidence,whereby the width and length of the focused line at the back focal planeof these rays can be suitably adjusted in relation to the size of thepixel and the pixel-separation in the pixel-array of the photodetector,or in the case of a spot-position sensitive large area sensor, inrelation to its area. As such, the number of pixels, or the area of thephotodetector, covered by the spot-intensity peak-width can be optimizedfor providing a high spot-position resolution, enabling a highsensitivity of measured wavelength of incidence.

As described in FIG. 13, the collimated beam passing the aperture isinternally reflected at 0′, passes lenses L3, L4, and L5, and grating T,reaching the detector plane at 0.

The above-described wavelength monitoring construction is illustrated inmore detail in FIG. 12, which shows a meridian section of ray bundlesreflected at a fixed angle at the sensor surface. While in theillustrated case, the dispersive element is a transmission grating, T,it may also be a prism. Rays at each specific wavelength λ1, λ2 and λ3,are collimated by the spherical objective, SO, in combination with thecylindrical elements, L3 and L4, diffracted by the grating, T, andsharply focused by another cylindrical lens, L5, to a line at meridionalpositions λ1, λ2, and λ3, on the detector, D, via folding mirrors, M1and M2.

The dispersive element may be in the form of a focusing reflectivegrating, positioned at M1 or M2, in a design replacing the equivalentlyfunctioning transmission grating T and focusing lens L5 in FIG. 12.

FIG. 13 shows a sagittal section of ray bundles reflected at differentparts of the sensor surface, sensor zone positions 4′, 5′, 6′, and 7′being sharply imaged by the spherical objective, SO, at sagittalpositions 4, 5, 6, and 7 at the detector plane, while rays reflected atsensor zone position 0′ also pass two cylindrical lens elements, L3 andL4, creating a collimated beam incident on the transmission grating, T.The light beam then passes a cylindrical lens, L5, which focuses thediffracted light in the meridional plane, but defocuses it slightly atthe ray-detector plane intersection at sagittal height 0. For the rayspassing only the spherical objective SO, the detector plane ispositioned at the real image plane of the lens system.

An alternative embodiment is shown in FIGS. 14 (meridional view) and 15(sagittal view), where the collimated beam is deflected by first andsecond mirrors, M1 and M2, and then passes the dispersive element, herea transmission grating, T, followed by the focusing lens, L5.

FIGS. 16 and 17 show an alternative embodiment of the optical biosensorsystem for ellipsometry. The specific ellipsometer components are shownas a typical ellipsometric set-up, and other ellipsometer configurationsare obvious to a person skilled in art.

With reference first to FIG. 16, the illustrated biosensor systemcomprises a light source, LS, and a collimator optics, CO, to produce aparallel beam. The latter passes an interference filter, I, and then, asa monochromatic beam, passes a first linear polarizer, P1. Thes-component of the this linearly polarized light is then retarded by aquarter-wave plate, Q, below called compensator, which creates anelliptically polarized light beam, impinging on a first flat scannermirror, SM1. Mirror SM1 deflects the beam onto a second scanning mirror,SM2, which in turn deflects the beam into a coupling prism, Pr (asbefore, grating coupling is also possible). The beam is totallyinternally reflected at the sensor interface side of the coupling prism,and then passes a second polarizer, P2, below called analyzer. A mainfirst part of the beam is directed into a first main part of anobjective consisting of a spherical objective, SO, producing a realimage on a first rectangular main area part detector array, D, of thelight intensity reflected from the sensor area.

In an alternative embodiment, the linear polarizer P1 followed by thecompensator are positioned between the mirror SM2 and the prism Pr. Fora suitable rotation of both the polarizer and analyzer, at a suitableset-up of orientation of the compensator, angle and wavelength ofincidence, the light reflected from a sensor zone can be extinguished.Upon a change in the refractive index or thickness of the sample in thezone, the extinction can be followed by scanning the angle and/orwavelength of incidence.

FIG. 17 shows a meridian view of the optical biosensor system, parallelto the view in FIG. 16. Here the ray path for a second, minor part ofthe beam is directed into a second, minor part of the objectiveconsisting of the spherical objective, SO, combined with two cylindricallenses, CL1 and CL2. This lens combination creates a projection of theserays so that the collimated beam reflected at different angles, hererepresented by beams a′, b′ and c′, within the scanned range is focusedonto a second linear minor part of the detector area, at a, b and c,respectively, this detector area being separated from the detector areaused for real image monitoring, so that each angle of reflectance willcorrespond to a specific detector position within this detector area.Thus, the above-mentioned second part of the objective has its backfocal plane positioned at the plane of the photodetector array.

The above described meridian bifocal imaging system in combination withan ellipsometer provides a simultaneous monitoring of both the positionof a reaction site within the sensor area (real imaging), and of thequantitative measure of the amount of reacting species at the site, viaat least one of the angle or wavelength of incidence, by use of the samedetector array.

From the above description of exemplary optical system designs embodyingthe present invention it is readily seen that monitoring the actualchange of at least one of the angle and wavelength of incidence via thebifocal imaging represents a considerable advantage in relation to theprior art in that the need to derive this change from any steeringsignal driving the angle scanner or scanning monochromator iseliminated. The scanner driver electronics and software thus just haveto provide a suitable dynamic range and scan speed for the angle orwavelength.

The above-described optical designs demonstrate the optical function ofthe present invention. It is, however, readily appreciated by a personskilled in the art, that these optical designs could be optimized toachieve a suitable performance and function of an apparatus constructedbased on these designs.

Also, while in the above described optical systems the bifocal (in themeridian plane) imaging system is based on lens optics, it is readilyunderstood that the imaging system may comprise diffractive opticalsurfaces for improving imaging quality, image plane flatness, and imageplane tilt, to match the needs of the photodetector array. Furthermore,diffractive optics may be the main component in the objective whichforms the above described real image and angular projection on twoseparate areas of the same detector array.

In the following, the different parts of apparatus embodying theabove-described optical functions will be described in more detail.

Thus, in accordance with the embodiments of FIGS. 1 to 10 describedabove, the optical construction employs an illumination systemcomprising a mainly monochromatic light source and a scanning planemirror system, an optical coupling component (plane sided prism orgrating) in optical contact with a sensing surface, an imaging systemcomprising a bifocal objective and a photodetector matrix array. Analgorithm and a computer program are provided which permit aninterpretation of the signals from a first main part of thephotodetector into a real image of the sensor pattern, and from a secondminor part of the photodetector into the angle of incidence of theprobing light. The angle of incidence at a specific parameter of thelight intensity, e.g., minimum or maximum, for each sensor zone may thusbe monitored simultaneously.

For example, the invention permits a beam having a diameter within therange of about 3 mm to 30 mm to be angularly or wavelength scanned andmonitored at a resolution of 0.0001 degrees, and 0.002 nm, respectively,within the range of angle or wavelength of interest, wherein the raysare collimated to a required degree (preferably within about 0.002degrees).

Illumination System

The illumination system creates a collimated beam at a suitablemonochromatic wavelength. In the angular scan mode of the invention, thebeam is scanned by a plane mirror system creating a collimated beam, theangle of incidence of which at the sensing surface is scanned while theilluminated area at the sensing surface is mainly fixed. In thewavelength scan mode, a wavelength dispersive device in combination withcollimator optics create a collimated beam which is incident at thesensing surface at a fixed angle.

The light incident on the total internal reflection interface may bep-polarized or consist of both p- and s-polarized components, i.e.,being suitably elliptically polarized.

The light source may be either mainly non-coherent, e.g., a lightemitting diode, a tungsten-halogen lamp, or mainly coherent, e.g., alaser diode.

In an apparatus based on angular scanning, the light source may beeither mainly monochromatic, e.g., a light emitting diode in combinationwith an interference filter, or a laser diode, or a light source capableof sequentially emitting a suitable number of wavelengths, one specificwavelength at each angular scan.

In an apparatus based on wavelength scanning, the light source may,e.g., be a white light source, such as a tungsten-halogen lamp, incombination with a scanning wavelength dispersive device. Such ascanning monochromator may be motorized to sequentially emit wavelengthswithin a wavelength range, which in combination with the collimatorcreates a collimated beam.

Angle Scanning Means

Fundamentally, scanning devices are light beam deflectors. Deflectorscan be categorized into reflective, refractive, and diffractive(acousto-optic scanners, holographic scanners). Scan patterns andscanning motions are inextricably interrelated involving both beamdisplacement and beam-displacement rate, and beam deflection andbeam-deflection rate. Scanning motions essentially fall into three basicmovements, rotational, oscillatory, and translational.

The scanning of the angle of incidence with time of a parallel lightbeam by utilizing a collimated beam deflecting element, may be createdby low inertia components, e.g., vibrating or rotating mirrors or amoving grating, or an inertialess acousto-optical deflector.

The beam deflecting system for an angularly scanned imaging consists ina preferred embodiment of mirrors, which as non-refractive optics doesnot need to be corrected for chromatic aberrations. In a preferred form,two oscillating or rotating inter-related plane mirrors are combinedwith a plane sided coupling prism, or a grating, which deflects acollimated beam during the angular scan of the beam so that the opticalaxis of the beam always intersects mainly the same point at the sensorsurface (to obtain reduced beam walking, i.e., improved stability of thelight intensity which improves the sensitivity of the instrument), andan imaging system designed to create a real image of the sensor surfacein combination with a simultaneous projection of the reflected lightinto an instant measure of the actual angle of incidence.

The incident angle for collimated beams should typically be scannedwithin an angular range of ±5°, with a detected angular resolution of≦0.0001°, corresponding to a refractive index resolution of 0.000001.For real-time monitoring, the angular scan should be rapid.

A preferred scanned angular interval is ≈70±6° at a wavelength of 820nm, alternatively, ≈77±8° at a wavelength of 660 nm.

The bifocal imaging system described above, may be combined with any ofthe numerous arrangements for angular scanning of a collimated incidentbeam available in the literature.

A preferred angular scanner comprising two plane mirrors, the turningmovements of which are interrelated via the steering electronics, hasalready been described in connection with FIGS. 1 and 2.

A first alternative scanning principle is the twin parallel-mirrordescribed in Harrick, N.J., Internal Reflection Spectroscopy, HarrickScientific Corp., 1987, New York, p. 185, and shown in FIG. 18 herein,however, after replacing the convex coupling lens in FIG. 18 with aprism as in the specifically described embodiments of the presentinvention.

A second alternative scanning principle is an optically optimized (i.e.,to obtain overlapping focal surfaces for the whole angular range) systemcomprising one plane mirror and focal surface flattening optics, inaccordance with the scanners described by Lenferink, A. T. M. et al.,Sensors and Actuators B, 3 (1991) p. 262, shown in FIG. 19 herein, andOda, K., et al., Optics Communications, Vol. 59, No. 5, 6, 1986, p. 362,shown in FIG. 20 herein.

Still another alternative scanning principle, equivalent to the previousone for focal surface flattening optics, consists of a plane mirror,SM1, scanning a focused beam from the illumination source, LS, along afocal surface of a concave mirror, Mc, creating an angle scannedcollimated beam, as shown in FIG. 21. This may also be described as thatthe illumination system includes one oscillating/or rotating planemirror in combination with a concave cylindrical mirror, which deflectsa focused beam during the angular scan of the beam so that its focalsurface overlaps the focal surface of the cylindrical mirror, therebycreating a scanning collimated beam.

Still another alternative scanning principle comprises a pivotallymoving illumination system, e.g., such as that described in WO 93/14392,and shown in FIG. 22 herein.

Still another possible scanning structure is an excentric rotatingpolygonic scanner, replacing the cooperating mirrors SM1 and SM2 inFIGS. 1 and 2. Typically, such a polygonic scanner consists of eightplanar mirrors mounted on the periphery of a rotating wheel.

Wavelength Scanning Means

When the reflectometry in the method and apparatus of the presentinvention is performed as a function of the incident wavelength at afixed angle, the light source is combined with a scanning monochromatorwhich in combination with the real imaging and image detectionsequentially creates a series of images, each of a specific wavelength.A smaller part of the same image detector (or a separate detector) isused for measuring the wavelength. For this dual detector function to bepossible, this smaller detector part is combined with a wavelengthdispersive element, which spatially separates light at differentwavelengths.

The incident wavelength should typically be scanned within a wavelengthrange of ±200 nm, with a detected wavelength resolution of ≦0.002 nm,corresponding to a refractive index resolution of 0.000001.

In its simplest form, the dispersive element consists of a prism. Due tothe refraction of the light in the prism, the rays must be deflected by,e.g., a reflective element, in order to reach the detector.

A grating spectrograph is similar to a prism spectrograph. The light tobe analyzed passes first through a combination of a slit and acollimating lens. It then reaches the reflecting or transmissiongrating, set with its grooves parallel to the slit, where light ofdifferent wavelengths is diffracted at different angles, and is for eachorder drawn out into a spectrum. A second lens focuses these diffractedrays onto the detector array. A grating with a high groove density perunit width may give a few orders, but their spectra are spread out muchwider than those formed by a lower density.

Generally, as is understood by the skilled person, the bifocal imagingsystem described above with the specific embodiments of the biosensorsystem of the invention may be combined with numerous arrangements forscanning monochromators available in the literature.

Sensor Surface and Opto-coupling to the Collimated Beam

In a preferred embodiment, the collimated light impinges on theinterface between sensor surface substrate and the actual sensor surfaceunder total internal reflection conditions. The substrate/sensor surfacemay be a separate exchangeable component consisting of a sensing layercoating a transparent substrate, e.g., of glass or plastic, that is inoptical contact with the optical coupling media described above. Thesubstrate should be matched in respect of refractive index to a couplingprism that transmits the light beams of the illumination system to thesensor surface. Alternatively, a plane side of the coupling prism may bethe substrate.

According to an alternative opto-coupling principle, the substrate maybe in the form of a grating on either the sensor surface substrateinterface, or on the opposite side to this interface on the substrate.

When, for example, the detection principle is based on SPR, the sensorsurface comprises a plasmon-active material, such as gold or silver. Inthe case of internal Brewster angle detection, on the other hand, thesensor surface is of a transparent material, i.e., there is no metalfilm.

The sensor surface part at the sensor surface plane that provides thelight for the detection of angle or wavelength should have a lowvariation in reflectance for the scanned angle/wavelength range. Avarying light intensity during the scan mode of the invention, e.g., ascaused by SPR also within this part of the detected light, is likely todisturb the accuracy of the angular determination. It is thereforepreferred to use for the angle/wavelength measurement a part of thesensor surface that is not brought in contact with the sample, e.g., apart in contact with flow cell material in case of a sensor surfacedocking system of the type described in the aforementioned U.S. Pat. No.5,313,264. Such a part may be located, e.g., at the flow cell wall, bythe software used in the instrument. Thereby, the light intensity of thefocused line on the detector will be insensitive to any lightinteraction with the sample during the angular or wavelength scan.

Alternatively, a purely reflecting part (no evanescent wave) of thesensor chip is used for the measurement of the incident light angle orwavelength to obtain a light beam intensity during the angular orwavelength scan that is not influenced by the sample.

Imaging System

Depending on the choice of microscopy monitoring or degree of large areamonitoring, the person skilled in art may design a suitable imagingsystem.

In the case of angular scanning, for each specific incident angle thereflected light is collected by an objective, which images a mainlystationarily illuminated sensor area. A possible marginal walk of theilluminated area is not a problem, provided that it is the stationarilyilluminated central part of the illuminated sensor surface that isimaged. The imaging system creates an image in the detector plane wherethe reflectance minima or maxima correspond to the local distribution ofrefractive index over the sample area. An optimum light intensity forimaging requires that the main part of the illuminating optical power islocalized to the stationarily illuminated and imaging part of the sensorsurface.

For microscopy applications, the sensor area is enlarged, typically20-40×. This image may be detected by a photodetector matrix, e.g., ofCCD (charged coupled device) camera type. Alternatively, this image maybe further enlarged by an ocular, typically 10-20×, and projected on thephotodetector matrix.

For large area applications, the sensor surface is generally reduced.Typically, a sensor area of, say, 3 cm×3 cm is reduced about 0.3×-0.6×on a conventional CCD-detector.

Two exemplary designs of imaging systems for use in the presentinvention are given below:

-   1. An objective of magnification 15× together with a photodetector    array with pixel dimensions 15×15 μm², and an array-area of 6×8 mm²,    may monitor a total sensor area of approximately 0.4×0.5 mm² with a    lateral resolution of 1×1 μm².-   2. An objective of magnification 0.2× with pixel dimensions 15×15    μm², and an array area of 6×8 mm², may monitor a total sensor area    of approximately 3×4 cm² with a lateral resolution of 75×75 μm².    Image Processing and Monitoring Software

An outline of the requirements on the computer software for the analysisand presentation of the monitored reaction zones is given below.

By the use of non-coherent light, the distribution of totally internallyreflected reflectance minima over the sensor surface may be detected inthe form of images synchronized to the incident angle of theilluminating light. The image reflectance data and image-related dataare processed in real-time in an image processing computer program toprovide, for example, a three-dimensional refractometric image with thequantified mass-distribution as a function of sensor surface coordinate.

A high time resolution between the refractometric images requires ashort angle-scanning period. A typical commercially interestingframe-rate range is of the order of 50-60 Hz. Each frame of detectedintensity corresponds to a specific angle or wavelength. Thus, duringone second a number of 50-60 intensity- and angle-data points are readfor each of the detector elements corresponding to a sensor zone.

To obtain a suitable mean value for a cluster of detector elementscovering a specific sensor zone, the image processing softwarecalculates a reflectance curve for the zone by first averaging theintensity of the chosen elements at each angle or wavelength, and thenplots this intensity versus the angle or wavelength. For example, eachzone may obtain one reflectance curve per second (i.e., the reflectancecurve rate is 1 Hz). A second algorithm calculates the angle orwavelength at the curve parameter correlated to the refractive indexwithin the sampled zone, normally, the minimum reflectance or centroidof the dip in the reflectance curve.

The higher the rate of the sample interaction with the sensor zone, thehigher angle and/or wavelength resolution is required, and the higherframe rate will be needed for the image processing.

The real image on a first main part of the matrix detector is read andstored by a so-called frame-grabbing program, while the position ofmaximum intensity, or alternatively, the centroid of the intensitycurve, for the angle- or wavelength-related beams focused on a secondminor part of the detector matrix is calculated and stored by a suitablealgorithm.

An exemplary measurement/calculation procedure based on the angular scanmode is described below.

First, an initial normalization of reflectance data is performed bymeasuring total reflectance during an angular scan without sampleaddition. This procedure may comprise the following steps:

a) Define for each sensor zone a cluster of pixels on the photodetectormatrix, including a center pixel and a selected number of neighboringpixels,

b) start a clock for the measurement process,

c) start driving an angle scan over a predetermined angular range(driving is, e.g., stepping or rotational motor control or moving coilcurrent for oscillating mirrors),

d) read from the 2-D image detector part of the photodetector matrixinto an image data memory, a sequence of raw data images for averaged2-D reflectance from the detector pixels corresponding to each sensorsurface zone,

e) simultaneously, read from the angle detector pixel row of thephotodetector to an angular data memory, a sequence of raw data for thereflectance intensity peak (pixel number, time),

f) calculate for each 2-D image the incident angle from the pixel numberof the respective intensity peak on the detector pixel row,

g) store the angle and time for the respective raw data images in animage/angle/time matrix,

h) calculate a normalizing matrix from the measured reflectance valuesof the 2-D image, the normalized reflectance being identical within thesensor zones for all angles, and

i) store a normalizing matrix (normalizing data, angle).

The system is now ready for monitoring sample surface concentrations ateach sensor zone by measuring reflectance minima during the angularscan. Measurements are performed first with only solvent at the surfaceand then with sample plus solvent. The measurement procedure maycomprise the following steps:

a) start a clock for the measurement process,

b) start driving an angle scan over a predetermined angular range,

c) read from the 2-D image detector part of the photodetector matrixinto an image data memory, a sequence of raw data for averaged 2-Dreflectance images from the detector pixels corresponding to each sensorsurface zone,

d) simultaneously read from the angle detector pixel row or rows of thephotodetector to an angular data memory, a sequence of raw data for thereflectance intensity peak (pixel number, time),

e) calculate for each 2-D image the incident angle from the pixel numberof the respective intensity peak on the detector pixel row,

f) store the angle and time for the respective images in animage/angle/time matrix,

g) calculate a normalized 2-D image from the raw data matrix of the 2-Dimage by correcting with the respective normalizing matrix,

h) store the angle and time for the respective normalized image in anormalized image/angle/time matrix,

i) select a specific sensor zone and the corresponding part of thenormalized image/angle/time matrix,

j) copy data for the angular dependence of the reflectance curve(normalized reflectance, angle),

k) calculate from the data in j) the angle and reflectance of thereflectance minimum,

l) calculate from the normalized reflectance matrix a medium time forthe angular scan ((start time +stop time)/2),

m) store a medium time for the reflectance minimum angle (in k)) and thespecific sensor zone in a reflectance minimum angle/sensor zone/mediumtime matrix,

n) calculate the reflectance minimum angle shift for the respectivesensor zone and time in relation to a reference angle for the sensorzone obtained before the sample reaction therewith,

o) calculate from the angle shift the internal surface concentration ofthe sensor zone,

p) store the internal surface concentration in a matrix (surfaceconcentration, medium time),

q) select the next specific sensor surface zone and calculate thesurface concentration as above,

r) calculate a differential surface concentration for selected referencezones,

s) store the relative surface concentration in a matrix (differentialsurface concentration, medium time),

t) present simultaneously in graphs or tables the internal and relativesurface concentrations, respectively, as a function of time for therespective surface zone,

u) start the driving of the next angular scan, and

v) repeat the above measuring procedure up to a final analysis time.

The driving of the angle may be continuous and the storing of the imageand angle data may be trigged by the angle data scanning over thepredetermined angular range. These stored image/angle/time-matrices areshifted into the calculation and presentation procedures at a ratedepending on the computer performance.

Instead of measuring and presenting surface concentrations, it is, ofcourse, possible to measure and present surface concentration changes,surface refractive indexes, surface refractive index changes, surfacethicknesses and surface thickness changes.

The amounts of sample species bound or adsorbed to the different sensorspots or subzones may be related to each other by analytical software.The time relation of the refractometric images makes it possible toobtain via further image data processing mass distribution kinetic datafor, e.g., specific sample binding/desorption, sample displacement alongthe sensor surface, or for the separation process.

The use of coherent light makes it possible to detect, in addition tothe distribution of total internal reflectance minima over the sensorsurface, also interferometric changes caused by locally changed massdistributions. Image data processing of the lateral movement pattern ofsuch interference bands may be used to obtain an increased sensitivityto lateral refractive index changes on the sensor surface.

The invention is, of course, not limited to the embodiments describedabove and shown in the drawings, but many modifications and changes maybe made within the scope of the general inventive concept as defined inthe following claims.

Experimental Procedure Example

The principle of the present invention was verified as demonstrated inFIGS. 24 a-24 d, by use of a pivotally moving illumination systemsimilar to the one described in FIG. 22, using a light emitting diode ofcenter-wavelength 766 nm, lenses and a sheet-polarizer providingp-polarized collimated beam of diameter 8 mm, an interference filter ofbandwidth 3 nm, an imaging embodiment according to FIGS. 3, 4, and 5,and a CCD-photodetector matrix connected to a video-recorder. Theobscuration, imaged as the dark right part in FIGS. 24 a-24 d coveringabout 25% of the image, is positioned at the interference filter I, FIG.23, decentered in relation to the beam cross-section, so that it coversabout 15% of the beam width.

FIGS. 24 a-24 d show a series of SPR-images of a sensor surface exposedto rinse solution in a flow cell, the sensor surface consisting ofsixteen zones of various refractive index (optical thickness), zone size0.5 mm×0.5 mm, each image at a specific angle of incidence increasing inthe order from a) to d). The lower the position of the focused spotwithin the angle-detecting area of the CCD, the larger is the angle ofincidence. The angle of incidence is 65.9° in FIG. 24 a, 66.4° in FIG.24 b, 66.9° in FIG. 24 c, and 67.4° in FIG. 24 d.

Since a change in the angle for SPR of 0.1° here corresponds to a changein the refractive index of 0.001 within the zone's reactive layer, theshift in refractive index is 0.005 between FIGS. 24 a and 24 b, 24 b and24 c, and 24 c and 24 d, respectively.

At SPR within a specific zone, detected as a minimum of light reflectedin the zone, the corresponding angle of incidence is a measure of therefractive index (optical thickness) of the zone. The larger the angleof incidence at SPR is, the larger is the optical thickness (orrefractive index at fixed thickness), which corresponds to a largersurface concentration of bound sample to a zone.

In FIG. 24 a the following zone-coordinates (row, column) show lightextinction due to SPR: (1,1), (1,2), (1,3), which the higher angle ofincidence in FIG. 24 b causes SPR in the zones: (2,1), (2,2), (3,1),(3,2), revealing a higher surface concentration in the latter zones. InFIG. 24 c, the zones of the sensor surface having yet higher surfaceconcentration of sample are revealed in zones (4,2) and (4,3). In FIG.24 d, the zone of the sensor surface having the highest surfaceconcentration of sample are revealed in zone (4,1).

As an example, the refractive index in zone (3,1) is 0.005 higher thanthe one for zone (1,1), while the refractive index in zone (4,2) is0.005 higher than the one for zone (2,1), and the refractive index inzone (4,1) is 0.015 higher than the one for zone (1,1).

The above-described results demonstrate the function of the presentinvention, an optical apparatus for multi-zone quantitative analysis ona sensor surface by use of simultaneous measuring of zone-data andangle-data on the same photodetector matrix. It is then readilyappreciated by a person skilled in the art, that a suitable evaluationcomputer program could provide a high-sensitive and fast determinationof the angle for SPR for specific sensor zones by combining a firstcomputer program for one-dimensional spot-position analysis with asecond computer program for two-dimensional intensity-pattern analysis.

Furthermore, it is readily appreciated by a person skilled in the art,that the invention also provides for measurement of zone-data on aphotodetector matrix and a simultaneous measurement of angle-data on aseparate detector.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An analytical system, comprising: a sensor unit having a sensingsurface with a number of individual zones, means for illuminating thesensing surface with a collimated beam of light, means for imagingreflected light from the illuminated sensing surface into an imageplane, means for repeatedly varying the incident angle and/or wavelengthof the light incident at the sensing surface over an angular and/orwavelength range, means for synchronized detection of images in theimage plane and incident angle and/or wavelength of light illuminatingthe sensing surface, and evaluation means for determining from therelationship between detected intensity of different parts of the imagesand incident light angle and/or wavelength, the optical thickness ofeach zone of the sensing surface, wherein the evaluation means furtherare arranged to determine the relative phase of p- and s-polarizedelectric field components of the reflected light.
 2. The systemaccording to claim 1, wherein the means for synchronized detectioncomprises integral photo-detector means.
 3. The system according toclaim 1, wherein the means for varying the incident light and/orwavelength comprise beam deflecting means to produce an angle-scannedcollimated illumination of the sensing surface.
 4. The system accordingto claim 1, wherein the sensing surface supports reactants capable ofbinding interaction with species in a sample.
 5. The system according toclaim 1, wherein the system comprises a flow cell in contact with thesensing surface to expose the sensing surface to a sample solution. 6.The system according to claim 1, wherein the collimated light beam iselliptically polarized.
 7. The system according to claim 1, wherein thesystem is based on total internal reflection versus angle and/orwavelength of incidence.
 8. The system according to claim 7, wherein thesystem is based on surface plasmon resonance.